The endothelium plays a central role in the control of vascular tone by producing vasoactive mediators like nitric oxide (NO). NO is generated from the amino acid L-arginine by the enzyme NO synthase (NOS), which requires NADPH and O2 and the cofactors FMN, FAD, heme, tetrahydrobiopterin and thiol. Among the five classes of NOS inhibitors which have been described are the flavoprotein binders like diphenyleneiodonium (DPI). DPI and its analogues are also inhibitors of the enzyme NADPH oxidase, which has recently been suggested as a possible oxygen sensor in several cell types. Inhibition of this enzyme by DPI irreversibly inhibits the hypoxic chemoreceptor response in the rat carotid body and blocks the effect of hypoxia on an outward K+ current in cultured pulmonary neuroepithelial bodies. We recently showed that DPI, like the arginine analogue and NOS inhibitor NG-nitro-L-arginine methyl ester (L -NAME), inhibits endothelium-dependent relaxation to acetylcholine (ACh) in isolated rat aortic rings (n = 10), which is mediated by NO (Circulation 1994;90:I-459). In addition, we have shown that both DPI and L- NAME produce an additional contraction of aortic rings partially preconstricted with prostaglandin (PG)F2beta. While the onset and magnitude of this contraction are similar for both NOS inhibitors (+145 plus minus 38% for L-NAME vs. +92 plum minus24% for DPI at 1 minute, P = NS), the L-NAME contraction is sustained throughout the period of exposure (+234 plum minus39% at 15 minutes) while that due to DPI is transient and followed by a more prolonged relaxation to a minimum tension of -27 angrstrons 19% at 15 minutes of continued exposure (n = 11, P <0.001 vs. L-NAME). This relaxation is endothelium- independent and may be mediated by a fall in vascular smooth muscle pHi (see below) which may alter myofilament sensitivity to Ca2+. Thus, while these two NOS inhibitors have similar effects on both endothelium-dependent vasodilation to ACh, the addition of DPI to PGF2beta-preconstricted rings reveals a vasodilation not found with L-NAME that may account for the different duration of the constriction each inhibitor initiates.